JP4710031B2 - Biochip substrate - Google Patents

Description

  The present invention relates to a biomolecule detection technique, and more particularly to a biochip substrate and a method for manufacturing a biochip substrate.

  The biochip is a general term for elements in which a probe that has a specific chemical reaction with a biological substance to be examined is fixed at a specific position on the chip surface. A DNA chip, which is a representative example of a biochip, is used to detect the type and amount of target DNA contained in blood or cell extracts. A DNA chip is obtained by arranging thousands to tens of thousands of probe DNAs, each of which is a single-stranded DNA having a known sequence, on a substrate such as a slide glass plate. When a test solution containing target DNA fluorescently labeled is supplied to the DNA chip, only the target DNA whose sequence is complementary to the probe DNA are bonded to each other by hydrogen bonding to form a double strand. Therefore, since the portion where the target DNA is immobilized develops fluorescence, the type and amount of the target DNA can be determined by measuring the fluorescence development position on the chip and the color development intensity. Therefore, when preparing a DNA chip to be used in this manner, it is necessary to fix a probe DNA having a predetermined sequence to a predetermined portion of the substrate surface.

  There are roughly two types of probe DNA immobilization methods. The first method is called a microarray method. This is a method in which probe DNA prepared by chemical synthesis in advance or extracted from a target living organism is dropped or printed in an array on a substrate and fixed. The second method uses a base of 4 types of bases, thymine (T: Thymine), adenine (A: Adenine), cytosine (C: Cytosine), and guanine (G: Guanine). This is a method of directly chemically synthesizing a probe DNA, which is a single-stranded DNA having a predetermined sequence based thereon. In the case of this second immobilization method, on the surface of the substrate used for chemical synthesis, a reaction region for proceeding with the synthesis reaction of the probe DNA for design purposes and a non-reactive region not involved in the synthesis reaction are clearly separated. It is necessary to be formed in the formed state.

In the case of a chip adopting the second fixing method, quartz (SiO ₂ ) or the like is used as the substrate material. In addition, a photolithography technique is used to form the reaction region and the non-reaction region separately. During chemical synthesis of probe DNA, single-stranded DNA is sequentially assembled by applying ultraviolet irradiation technology to the activation treatment of single-stranded DNA in the reaction region. In this chip, about 200,000 spots of reaction areas with a size of about 20 μm square are formed on a single substrate, and about 2 million probes of the same type are formed in one spot. DNA is fixed. Since the spots are formed at a high density on the chip, a very large number of target DNAs can be examined in one test.

  Another proposed chip is an array plate. The array plate is manufactured as follows. First, the surface of a silicon (Si) substrate is reacted with fluoroalkylsilane, and a hydrophobic fluoroalkylsiloxane thin film is once formed there. Next, the thin film is removed in a predetermined plane pattern, the surface of the Si substrate is exposed at the portion where the thin film is removed, and finally, the exposed surface is reacted with hydroxysilane or alkylsilane to thereby form an OH group on the exposed surface. (For example, refer to Patent Document 1). Therefore, in the case of an array plate, there are a site of a hydrophobic thin film having a large surface tension and a site having a hydrophilic OH group on the surface of the Si substrate. Therefore, the former functions as a non-reactive region and the latter functions as a reactive region with respect to a biological substance. When using the array plate, the synthesis reaction proceeds at the hydrophilic site, and then the test solution is supplied thereto. The test liquid is secured in the hydrophilic site by the large surface tension of the hydrophobic thin film located around the hydrophilic site. However, in the case of an array plate, since there is virtually no difference in height between the reaction region and the non-reaction region, it can be said that the stability is insufficient in terms of securing the supplied test solution and is difficult to use. In addition, the formation of the reaction region and the non-reaction region both depend on the chemical reaction between the Si surface and other chemical substances, and it cannot be said that the reaction proceeds in 100% yield. The boundaries may be ambiguous. Furthermore, there is a problem that the hydrophobic thin film and the hydrophilic site are easily damaged.

  In terms of securing the test solution, a substrate provided with a plurality of wells having a structure capable of storing and holding the test solution is more suitable than the array plate having the above-described structure. As such a substrate, a substrate used for direct synthesis of probe DNA and chemical synthesis of probe DNA using the same are disclosed (for example, see Patent Document 2). As shown in FIG. 61, the substrate A has a plurality of wells 2 formed in a predetermined arrangement on one surface of a semiconductor substrate 1 made of Si or the like. The diameter of the well 2 is about 1 to 1000 μm and the depth is about 1 to 500 μm, and this functions as a reaction region for chemically synthesizing the probe DNA. The surface portion other than the well is a non-reactive region. This substrate A is manufactured as follows.

Step a ₁ : Applying a photolithography technique and an etching technique to one surface 1a of the semiconductor substrate 1 to form a well 2A, thereby producing an intermediate A ₁ as shown in FIG.

Step a ₂ : For example, thermal oxidation treatment is applied to the surface 1 _a of the semiconductor substrate 1a and the surface (bottom surface and side surface) of the well 2A in the intermediate A _1, and only the surface layer portion of these surfaces is shown in FIG. An intermediate A ₂ converted to a silicon oxide (SiO 2) layer 3 having a thickness of about 0.5 μm is manufactured.

Step a ₃ : The surface of the Si oxide layer 3 of the intermediate A ₂ is subjected to silanization treatment. Specifically, after applying a brown process using sodium hydroxide (NaOH) to treat the surface of the Si oxide layer 3 with an alkali, for example, an epoxysilane type functionalized silane treating agent. Process with. Further, the silane treatment agent is linked and the epoxy resin is hydrolyzed. As a result, Si oxide layer 3 of the silane coupling layer 4 on the surface is formed, the intermediate A _3, as shown in Figure 64 is obtained. Since this intermediate A ₃ has OH groups of silanes on the entire surface thereof, the entire surface is in a state capable of reacting with the DNA phosphoramidite.

Step a _4: By applying the phosphoramidite method to the surface of the intermediate A _3, by combining the single-stranded DNA of a length of about 5 base using DNA phosphoramidite T (thymine), a silane coupling layer A spacer layer 5 made of an oligonucleotide spacer (5T) is formed on ₄ to produce an intermediate A ₄ having wells as shown in FIG. The end of the synthesized oligonucleotide spacer (5T) is protected with dimethoxytrityl (DMT). This intermediate A ₄ is in a state where the entire surface is covered with a spacer layer 5 of an oligonucleotide spacer (5T) protected with DMT. Therefore, if the end of the oligonucleotide spacer is activated by removing (detritylation) DMT that protects the end, it is possible to synthesize the probe DNA there. That is, in the case of the intermediate A _4, at the time of creation of the DNA chip, the entire surface of the spacer layer 5 of oligonucleotides spacer (5T) functions as a reaction region. However, in this state, the well does not satisfy the requirement as a substrate for probe DNA synthesis that the well must be separated as a reaction region and the other part as a non-reaction region. Therefore, the spacer layer 5 of the intermediate A ₄ needs to be treated so that the well portion remains as a reaction region and the other portion is converted into a non-reaction region. The process is an inactivation process (Capping) performed in the next process.

Step a ₅ : As shown in FIG. 66, the resin droplet 6 is filled only in the well of the intermediate A ₄ , and the inactivation treatment (Capping) of the spacer layer 5 is performed in that state. Specifically, the spacer layer 5 of the oligonucleotide spacer (5T) is detritylated to activate the end of the oligonucleotide spacer (5T), and then trichloroacetic acid, acetic anhydride, dimethylaminopyridine, etc. Is used to block and inactivate the activated end of the oligonucleotide spacer (5T) to convert the spacer layer 5 into the inactive spacer layer 5a. Thereafter, the resin droplet 6 filled in the well is dissolved and removed using an organic solvent such as tetrahydrofuran to expose the spacer layer 5 in the well. Since the well is filled with resin droplets during the inactivation process, the spacer layer 5 made of the oligonucleotide spacer (5T) in the well can react without being subjected to the inactivation process. The state is maintained. However, the oligonucleotide spacer (5T) in the portion other than the well is inactivated so that no synthesis reaction occurs. In this way, the substrate A ₆ on which the probe DNA having the cross-sectional structure shown in FIG. 67 is synthesized is manufactured.

In the substrate A _6, well 2 is formed at a predetermined pattern is recessing portion on the surface of the semiconductor substrate. A Si oxide layer 3 is formed on the entire surface of the semiconductor substrate 1, and a silane coupling layer 4 is formed on the entire surface of the Si oxide layer 3. On the bottom surface 2a and side surface 2b of the well 2, a spacer layer 5 consisting of an oligonucleotide spacer (5T) whose end is protected by DMT is exposed, and this portion is a reaction region for synthesizing probe DNA. It has become. However, the inactive spacer layer 5a is an inactivated portion, which is a non-reactive region. When a DNA chip is prepared by the phosphoramidite method using this substrate A, the chemical synthesis of the probe DNA proceeds in the well 2 region.

However, this substrate A ₆ has the following problems. The first problem is that in the case of the substrate A ₆ , the boundary between the reaction region and the non-reaction region is determined by the filling state of the resin droplets in step a ₅ . In general, filling of resin droplets into wells is performed using a piezo injector, but the filling amount is a very small amount of the order of pl to μl. Therefore, in step a _5, sometimes the resin droplet filling quantity is too large is not completely filled or overflows, the wells too small to reverse from the well. In the former case, the peripheral surface of the well is also covered with the overflowing resin droplets. Therefore, even at the time of the next inactivation treatment, the peripheral surface described above remains in a state in which the synthesis reaction can be performed without being inactivated. Therefore, probe DNA is chemically synthesized on the peripheral surface of the well when a DNA chip is produced. For this reason, when the target DNA is examined using the prepared DNA chip, fluorescent coloring occurs not only in the well portion but also in the peripheral portion of the well where the resin droplet overflows. As a result, accurate reading of the fluorescent label is inhibited.

Further, when the filling amount of the resin droplets is insufficient, the thickness of the resin droplets covering the surface inside the well is reduced, so that the resin droplets are easily eroded by the acid solution used during the inactivation process. As a result, the oligonucleotide spacer (5T) layer located in the well may be partially inactivated. Therefore, the probe DNA may not be sufficiently chemically synthesized inside the well when the DNA chip is produced. As a result, the portion of the well may not develop fluorescence with sufficient intensity when the target DNA is examined. Furthermore, when step a deactivated in ₅ (Capping) is insufficient, again, when using the DNA chip prepared not only part of the well, also fluorescent insufficient partially deactivated is Therefore, background noise is likely to occur. Further, in step a _1, when forming the well 2A, because the depth will come determined by the length of etching time, if not correctly performed the time management during the etching process, accurate in line with the design criteria A well with a sufficient depth may not be formed.
Japanese National Publication No. 9-500568 Special Table 2002-537869

  The present invention provides a biochip substrate capable of reducing background noise and a method for manufacturing a biochip substrate.

In order to achieve the above object, the first feature of the present invention is: (a) a silicon layer, (b) a silicon oxide layer disposed on the silicon layer, and (c) disposed on the silicon oxide layer. is provided with a non-reactive layer in which a plurality of wells is provided which reaches the silicon oxide layer, and (d) a biological material disposed on the surface of the silicon oxide layer exposed through the wells, non-reactive layer is summarized in that a substrate for a biochip comprising at least silicon.
The biochip substrate is preferably manufactured from an SOI (Silicon on Insulator) substrate.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the biochip board | substrate and biochip board | substrate which can reduce background noise can be provided.

  Embodiments of the present invention will be described below. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, the drawings are schematic. Therefore, specific dimensions and the like should be determined in light of the following description. Moreover, it is a matter of course that portions having different dimensional relationships and ratios are included between the drawings.

(First embodiment)
The biochip substrate according to the first embodiment is provided with a reaction region where a reaction for assembling a probe DNA using a DNA phosphoramidite can be performed and a non-reaction region where the reaction is not performed. Here, in the conventional substrate A shown in FIG. 67, the non-reactive region is formed by the inactivation process (Capping). In contrast, in the biochip substrate according to the first embodiment, the reaction region is formed of a reactive material, and the other regions are reliably formed of a material that is inert to the reaction. Therefore, there is a feature that the reaction region and the non-reaction region are clearly distinguished without performing surface inactivation treatment such as capping.

First, an example of the biochip substrate B _{0 according to} the first embodiment is shown in FIG. The biochip substrate B ₀ has a plate-like body having a sandwich structure in which a Si oxide layer 132 is interposed between two semiconductor layers 131 made of Si or the like. Here, a well 13A that reaches the surface 132a of the Si oxide layer 132 is provided in one semiconductor layer 131 in a predetermined arrangement pattern. Therefore, in the case of the biochip substrate B ₀ , the surface 132a of the Si oxide layer 132 is exposed only on the bottom surface of the well 13A, and the surfaces of the other portions are all Si. Therefore, only the surface of the Si oxide layer 132 exposed through the well 13A functions as a reaction region for synthesizing, for example, single-stranded DNA, and all other surfaces function as non-reaction regions.

FIG. 2 shows an example B ₁ of another substrate manufactured using the biochip substrate B ₀ described above. The substrate B ₁ represents a silane coupling layer 14 only on the bottom surface of the well 13A in the substrate B ₀ for the biochip is formed. On the silane coupling layer 14, a spacer layer 135 made of an oligonucleotide spacer (5T) obtained by continuously synthesizing DNA phosphoramidite T in five stages is disposed. In this substrate B _1, only the bottom surface 135a of the wells 13A, the spacer layer 135 of an oligonucleotide spacer that allows the probe DNA synthesis (5T) and are arranged, the surface 131a of the side surface 135b and the semiconductor layer 131 of wells 13A Both remain Si. Therefore, in the substrate B _1, only the bottom surface of the well 13A functions as a reaction region of the probe DNA synthesis, other parts such as the side and the substrate surface of the well 13A is inactivation of the surface by Capping treatment or the like is performed Even if not, it itself is a non-reactive region for the chemical synthesis of probe DNA. Incidentally, in the substrate B _1, silane coupling layer 14 has been arranged to carry out the synthesis of the spacer layer 135 consisting of an oligonucleotide spacer (5T) efficiently, it is not always necessary. A spacer layer 135 made of an oligonucleotide spacer (5T) may be directly synthesized on the surface 132a of the Si oxide layer 132.

The substrate B ₁ represents may be prepared as follows. First, as shown in FIG. 3, a plate-like body having a structure in which a Si oxide layer 132 is sandwiched between two semiconductor layers 131 is prepared. At this time, the thickness of one semiconductor layer 131 is substantially the same as the depth of the well to be formed. As such a plate-like body, for example, an SOI (Silicon on Insulator) wafer commercially available from Shin-Etsu Semiconductor Co., Ltd. is suitable.

  Next, as shown in FIG. 4, the surface 131a of one semiconductor layer 131 is entirely covered with a resist 16, and a mask having an opening having the same diameter as the well to be formed is disposed thereon, and then irradiated with ultraviolet rays. Further, the mask is removed and the whole is developed. As a result, as shown in FIG. 5, an opening having the same diameter as the well to be formed is formed in the resist 16, and the surface 131a of the semiconductor layer 131 is exposed therefrom.

  Next, using the resist 16 as a mask, the semiconductor layer 131 is etched using an Si etchant to form a well 13A that reaches the surface 132a of the Si oxide layer 132. The etching action is automatically stopped when the etching of the semiconductor layer proceeds and reaches the surface 132a of the Si oxide layer 132. As a result, as shown in FIG. 6, the surface 132a of the Si oxide layer 132 is exposed only at the bottom surface of the well 13A, and a member made entirely of Si is obtained on the other surface portions. The well 13A in this member has substantially the same diameter and depth as the well to be formed.

Next, silanization treatment is performed. Since the silanization reaction proceeds only on the surface of the Si oxide layer, as shown in FIG. 7, the silane coupling layer 14 is formed only on the surface 132a of the Si oxide layer 132 exposed in the well 13A. Finally, by applying the phosphoramidite method, DNA phosphoramidite T is reacted with the silane coupling layer 14 to form a spacer layer 135 composed of an oligonucleotide spacer (5T) having a single-stranded DNA composed of 5mer T. Thus, the substrate B ₁ shown in FIG. 2 is completed. In this process, since the DNA phosphoramidite T is covalently bonded only to the OH group of the silane coupling layer 14, the synthesis reaction proceeds only on the surface 132a of the Si oxide layer 132 exposed by the well 13A. Here, since the DNA phosphoramidite T does not react with other parts exposed by Si, it is not necessary to perform inactivation treatment (Capping) as in the case of the conventional substrate A.

When the biochip substrate B _{0 according} to the first embodiment is compared with the manufacturing process of the conventional substrate A described above, the following is clear. When first conventional substrate A, it is necessary that the surface of the Si wafer in the above-mentioned step a ₂ to form a Si oxide layer by thermally oxidizing the substrate for the biochip according to a first embodiment B If ₀ is the step a ₂ becomes unnecessary. In the case of a conventional substrate A, the filling of the resin droplets to the described process a ₅ in the wells, inactivation treatment (Capping), and a series of operation of dissolution and removal of the resin droplets was essential. In contrast, in the case of the biochip substrate B _{0 according} to the first embodiment, these operations are completely unnecessary. Therefore, in the case of the biochip substrate B ₀ according to the first embodiment, the problem that has occurred due to the excessive and insufficient filling amount of the resin droplets is solved, and the boundary between the reaction region and the non-reaction region becomes clear, and The reaction area is only the bottom surface of the well 13A formed by applying the lithography technique and the etching technique, and all other parts are non-reaction areas. For this reason, the accuracy of the fluorescent labeling when using a DNA chip prepared using the same is increased, and background noise is not generated.

Further, in the case of the conventional substrate A, in order to form a well having a depth conforming to design criteria, it was necessary to accurately manage etching time when etching steps a _1. On the other hand, in the case of the biochip substrate B _{0 according} to the first embodiment, the etching action for digging the semiconductor layer 131 is automatically stopped when the surface of the Si oxide layer is reached. Since the depth of the formed well 13A is uniquely determined by the thickness of the semiconductor layer 131 used, the depth of the formed well 13A becomes extremely accurate.

Another example B ₂ of the substrate shown in FIG. In the substrate B _2, for example, on only one surface portion with thermal oxidation of the Si oxide layer 141a of the semiconductor substrate 141 made of Si or the like that is a Si oxide layer 141a, for example as DNA phosphoramidite T A non-reactive layer 142 having a predetermined thickness made of a material that does not react with various biological substances and is not eroded by various organic solvents, acid solutions, alkaline solutions, and the like used during DNA synthesis is disposed. In the thickness direction of the non-reactive layer 142, wells 23 having a predetermined diameter are provided in a predetermined arrangement pattern up to the surface 141b of the Si oxide layer 141a. On the surface 141b of the Si oxide layer 141a, a spacer layer 135 made of an oligonucleotide spacer (5T) having a single strand DNA consisting of 5 mer by reacting with the silane coupling layer 14 and DNA phosphoramidite T is arranged in this order. Is arranged in. For the substrate B _2, constitutes a reaction zone exposed surface of the spacer layer 135 consisting of an oligonucleotide spacer of only the bottom surface of the well 23 is possible reactions with DNA phosphoramidite T (5T), the other surface Are not inactivated, but are all non-reactive regions made of a material that does not react with DNA phosphoramidite T.

  Here, examples of the material of the non-reactive layer 142 that forms the non-reactive region include single crystal silicon, metals such as platinum that are difficult to form oxides, nitrides such as silicon nitride, polyethylene, and polystyrene. Examples thereof include plastics that do not have a reactive functional group. The non-reactive layer 142 is formed by a method such as direct bonding of the above-described material member to the Si oxide layer, vacuum deposition or CVD of the above material on the surface of the Si oxide layer, or vapor phase polymerization using a monomer as a raw material. Etc. can be formed. The well 23 can be formed by applying, for example, a photolithography technique and an etching technique.

Another example B ₃ of the substrate shown in FIG. The substrate B ₃ is the surface of the glass plate 151, the same non-reactive layer 142 in the case of the substrate B ₂ is disposed in the thickness direction of the layer, the well 152 extending to the surface 151a of the glass plate 151 is provided The silane coupling layer 14 and the spacer layer 135 made of the oligonucleotide spacer (5T) are arranged only on the surface 151a of the glass plate exposed from the bottom surface of the well 152.

For the substrate B _3, the surface of the oligonucleotide spacer (5T) layer is exposed only on the bottom surface 151a of the well 152, which constitutes the reaction zone, the surface of the side surface and the non-reactive layer 142 of wells, inactivated All are non-reactive areas without any treatment.

Another example B ₄ of the substrate shown in FIG. 10. This substrate B ₄ does not react with the DNA phosphoramidite T, and from the surface 161a of the plate-like body 161 made of a single material that is not eroded by an organic solvent, acid solution, alkali solution, etc. used at the time of DNA synthesis, toward the inside. A well 162 having a predetermined diameter and depth is provided. A Si oxide layer 163 is disposed on the bottom surface 161a of the well 162, and a spacer layer 135 composed of the silane coupling layer 14 and the oligonucleotide spacer (5T) is sequentially disposed on the Si oxide layer. In the case of this substrate B ₄ as well, the part that can react with the DNA phosphoramidite T is only the spacer layer 135 made of an oligonucleotide spacer located on the bottom of the well, and the other part is a non-reactive region. Examples of the single material constituting the non-reactive region include Si and nitrides such as silicon nitride. When a plate-like body made of Si is used, the above-described well can be formed by applying, for example, dry etching such as reactive ion etching or ion milling, or wet etching.

  By applying the SIMOX (Separation by IMplanted Oxygen) method, in which oxygen ions are ion-implanted from the surface of the Si plate to the inside, a Si oxide layer is placed inside the Si plate at a certain depth from the surface. Then, a well may be formed from the surface of the Si plate to the Si oxide layer. In addition, after oxidizing the surface of the Si plate, openings are arranged in a predetermined pattern by applying photolithography and etching techniques to the Si oxide layer on the surface, and then exposed to the openings. Oxygen ions are implanted from the surface of the Si by the SIMOX method described above, and then the Si oxide layer on the surface is removed by etching, so that the surface of the Si plate is directly under the opening described above. An isolation structure of the Si oxide layer can be formed at a depth position. Then, a well may be formed from the surface of the Si plate to the Si oxide layer at the position of the opening.

Next, a step of creating a biochip using a substrate for biochips present invention, a substrate B ₁ shown in FIG. 2, an example of the case of creating a DNA chip probe DNA is fixed by the phosphoramidite method I will explain.

First, a substrate B _1. Substrate B ₁ represents, as shown schematically in Figure 11, all of the well 13 that are arranged in an array in the substrate B ₁ represents only its bottom surface, an oligonucleotide spacer end protected with DMT (5T The spacer layer 135 is fixed.

Step b ₁ : Resin masking is performed on the well. Specifically, out of the wells to be arranged, for example, wells that are chemically synthesized with DNA phosphoramidite C: (Cytosine) are excluded, and all the other wells are filled with the resin droplet 6. As shown in FIG. 12, the oligonucleotide spacer (5T) in the well filled with the resin droplet 6 is sealed with the resin, but the oligonucleotide spacer (5T) in the well not filled with the resin droplet 6 ) Indicates that the terminal DMT is removable.

Step b ₂ : An acid solution such as trichloroacetic acid is uniformly supplied to the entire upper surface of the substrate to perform a detritilation process (Detritilation). As a result, as shown in FIG. 13, in the well not filled with the resin droplet, the DMT of the oligonucleotide spacer (5T) is detached and the oligonucleotide spacer (5T) is activated. Thus, among the wells provided on the substrate B _1, only the oligonucleotide spacer in a particular well (not filled with the resin droplets wells) (5T) is capable of reacting conditions with DNA phosphoramidite C.

Step b _3: the surface of the substrate, by supplying an organic solvent to dissolve and remove the resin droplets is filled into the wells. As a result, as shown in FIG. 14, an activated oligonucleotide spacer (5T) and an oligonucleotide spacer (5T) whose ends are protected with DMT are exposed on the bottom surface of the well arranged on the substrate. To do.

Step b _4: the entire surface of the substrate, end evenly supplied reagents protected DNA phosphoramidites C with DMT performing DNA synthesis (Coupling). As a result, as shown in FIG. 15, in a specific well, the synthesis reaction proceeds between the activated oligonucleotide spacer (5T) and the supplied DNA phosphoramidite C, and both are chemically bonded, Oligonucleotides extend 1mer. The end of the extended oligonucleotide is protected with DMT. On the other hand, in the remaining wells, the exposed oligonucleotide spacer (5T) is inactive because the ends are protected with DMT. Therefore, the synthesis reaction does not proceed with the supplied DNA phosphoramidite C.

Thus, by advancing the DNA synthesis reaction using steps b ₁ ~ Step b ₄ as one cycle, the oligonucleotide only wells not filled with resin droplets is 1mer extended above 1 cycle, filled with resin droplets In the well, the state before the start of the synthesis reaction is maintained. Since there are four types of DNA phosphoramidites to be reacted, T, A, C, and G, the above cycle may be performed four times in order to extend the oligonucleotide fixed to the well by 1 mer. In this manner, only the bottom surface of the well substrate B _1, it is possible to fix the probe DNA sequence based on the design diagram.

When creating a DNA chip using the substrate B _1, DNA synthesis reaction proceeds only at a bottom surface of the well, the other part is not at all involved in the synthesis reaction. Thus, for example, even a resin droplets filling in step b ₁ is overflowing if the well, as long as it does not flow into the adjacent wells is independent of the DNA synthesis reaction, adversely any adverse effect upon the fluorescent identification Absent. Further, it is not necessary to perform the inactivation process (Capping) which is performed in the case of the conventional substrate A.

In the above description, all of the substrates B ₁ , B ₂ , B ₃ , and B ₄ enable a probe DNA synthesis reaction by forming a silane layer on the surface of the Si oxide layer of the substrate B _0. The present invention relates to a DNA chip substrate. However, the biochip substrate according to the first embodiment is not limited to this, and specifically binds to the biological substance to be tested as a material constituting the bottom surface (reaction region) of the well. Various chips can be manufactured by selecting a material capable of fixing a biological substance.

For example, by immobilizing various linker components that specifically bind to the biological material to be tested on the surface of the Si oxide layer of the basic structure substrate B ₀ shown in FIG. 1, the portion reacts with the biological material. Can be specialized in the area. In that case, the other part of the substrate becomes a non-reactive region for the biological material, and the boundary between the two regions is fractionated.

For example, if a linker component such as a silane coupling agent such as aminopropylmethoxysilane or a functional group such as an epoxy group, a tosyl group, an activated carboxyl group, an amino group, a thiol group, or a bromoatamide group is used. In addition, a biological substance having an amino group, a thiol group, a hydroxyl group, a carboxyl group, a bromoatamide group or the like as a terminal can be immobilized on the surface of the Si oxide layer of the substrate B ₀ via a linker component.

  In addition, if a double-stranded DNA, protein, peptide, sugar chain, RNA-protein complex, or carbohydrate-protein complex is used as a biological material, a transcription factor that recognizes and binds to a specific base sequence of the double-stranded DNA. A chip for detecting a group, a chip for peptide detection, a chip for protein detection, a chip for sugar chain detection, a chip for digesting proteins, and the like can be produced.

(Second embodiment)
A biochip substrate B ₁ having the structure shown in FIG. 2 was finally manufactured through the steps shown in FIGS. 3 to 7 using an SOI wafer as a starting material. The specifications of this biochip substrate B ₁ are as follows. The size is 1 cm × 1 cm, the shape of the well is 300 μm in diameter, and 20 μm in depth. Nine vertical and 14 horizontal wells are formed on the substrate. The silane coupling layer is formed using 5,6-epoxytriethoxysilane, and the oligonucleotide spacer layer is formed by a phosphoramidite method using a DNA synthesis reagent. Using this biochip substrate B ₁ , a phosphoramidite method is applied to synthesize probe DNA having the following sequence in the well to produce a DNA chip.

1. 3'-ATCTCACACGTCAAATAG-5 '
2. 3'-ATCTCACTCAAATAG-5 '
3. 3'-ATCTCACGCAAATAG-5 '
4. 3'-ATCTCACCCAAATAG-5 '
5. 3'-ATCTCACACAAATAG-5 '
6. 3'-ATCTCACCAAATAG-5 '
FIG. 16 shows the results of a detection test of fluorescently labeled target DNA using this DNA chip. Here, since the fluorescence intensity of the spot of the probe 4 is the highest, it can be seen that the sequence of the target DNA is 5′-TAGAGTGGGTTTATC-3 ′. In all of probes 2, 3 and 5, the base located at the center of the sequence is mismatched. Probe 1 has a too long base sequence, and probe 6 is too short to be mismatched.

  For comparison, the substrate A having the structure shown in FIG. 67 is manufactured by performing a deactivation process (Capping) in accordance with the method disclosed in JP-T-2002-537869. Then, using this substrate A, in the same manner as the DNA chip according to the second embodiment, a probe chip having six types of sequences was transplanted to create a DNA chip, and the results of the target DNA detection test are shown in FIG. Shown in Also in this case, the fluorescence intensity at the spot of the probe 4 is the highest, and it can be seen that the target DNA sequence is 5'-TAGAGTGGGTTTATC-3 '. However, as is clear from FIG. 17, in the DNA chip produced from the conventional substrate A, the periphery of each spot is also fluorescently colored. For this reason, background noise occurs, and the boundary between the light emission spot and the substrate surface is unclear. This is a result of the resin droplets overflowing from the well to the periphery thereof when filling the wells with the resin droplets during the manufacture of the substrate, and the portion was not inactivated. Further, in the case of the spot of the probe 4, the fluorescent color development at the center of the spot is weak. This is because the filling amount of resin droplets was too small. However, the overall fluorescence intensity is high because the probe DNA transplanted here is complementary to the target DNA.

In contrast to this conventional substrate A, the DNA chip produced from the biochip substrate B _{1 of} the second embodiment has a very clear boundary between the light emission spot and the substrate surface, and the occurrence of background noise is It may be said that it is substantially absent. Moreover, the fluorescence intensity in each light emission spot is also homogenized, and the measurement data is stable.

(Third embodiment)
The biochip substrate according to the third embodiment of the present invention is disposed on the semiconductor substrate 15 and the semiconductor substrate 15, as shown in FIG. 19 which is a cross-sectional view seen from the AA direction of FIG. 18 and FIG. A first layer 13 made of silicon oxide (SiO ₂ ) and capable of introducing a hydroxyl (-OH) group on the surface, arranged on the first layer 13, and a plurality of wells 41a reaching the first layer 13 , 41b, 41c, 41d, 41e, 41f, 41g, 41h, 41i, 42a, 42b, 42c, 42d, 42e, 42f, 42g, 42h, 42i, 43a, 43b, 43c, 43d, 43e, 43f, 43g, 43h , 43i, 44a, 44b, 44c, 44d, 44e, 44f, 44g, 45a, 45b, 45c, 45d, 45e, 45f, 45g, 46a, 46b, 46c, 46d, 46e, 46f, 46g, 47a, 47b, 47c , 47d, 47e, 47f, 47g, 47h, 47i, 48a, 48b, 48c, 48d, 48e, 48f, 48g, 48h, 48i, 49a, 49b, 49c, 49d, 49e, 49f, 49g, 49h, 49i It has a second layer 11. The second layer 11 is made of a material different from that of the first layer 13, and is made of a material such as crystalline silicon (Si) that is less likely to introduce a hydroxyl (—OH) group on the surface than the second layer 11. The first layer 13 has higher hydrophilicity than the second layer 11. Therefore, the first layer 13 is easily hydroxylated as compared with the second layer 11.

  Further, the biochip substrate shown in FIG. 18 includes a plurality of wells 41a to 41i, 42a to 42i, 43a to 43i, 44a to 44g, 45a to 45g, 46a to 46g, 47a to 47i, 48a to 48i, 49a to 49i. A plurality of flow paths 31a, 31b, 31c, 31d, 31e, 31f, 31g, 31h, 31i, 32a, 32b, 32c, 32d, 32e, 32f, 32g, which are connected to each other, 32h, 32i, 33a, 33b, 33c, 33d, 33e, 33f, 33g, 33h, 33i, 34a, 34b, 34c, 34d, 34e, 34f, 34g, 35a, 35b, 35c, 35d, 35e, 35f, 35g, 36a, 36b, 36c, 36d, 36e, 36f, 36g, 37a, 37b, 37c, 37d, 37e, 37f, 37g, 37h, 37i, 38a, 38b, 38c, 38d, 38e, 38f, 38g, 38h, 38i, 39a, 39b, 39c, 39d, 39e, 39f, 39g, 39h. The flow path 31a is a groove connecting the well 41a and the well 41b, as shown in FIG. 20 which is a cross-sectional view seen from the BB direction in FIG. 19 and FIG. Each of the plurality of other flow paths 31b to 31i, 32a to 32i, 33a to 33i, 34a to 34g, 35a to 35g, 36a to 36g, 37a to 37i, 38a to 38i, and 39a to 39h are also a plurality of wells 41b to 41i, 42a-42i, 43a-43i, 44a-44g, 45a-45g, 46a-46g, 47a-47i, 48a-48i, 49a-49i are grooves provided in the second layer 11 . Each of the plurality of wells 41a to 49i can be filled with the same solution by the plurality of flow paths 31a to 39h.

  As shown in FIG. 19, the surface of the first layer 13 exposed through each of the plurality of wells 41a to 41i has a biological material layer 91a, 91b, 91c, 91d, 91e, 91f, 91g, 91h, 91i is arranged. In each of the biological material layers 91a to 91i, each functional group of a plurality of biomolecules such as a plurality of deoxyribonucleic acids (DNA), a plurality of ribonucleic acids (RNA), a plurality of peptide nucleic acids (PNA), or a plurality of proteins However, as shown in FIG. 21, it is covalently bonded to a hydroxyl group (—OH) group on the surface of the first layer 13. When the biomolecule is DNA, RNA, or PNA, each sequence is designed to be complementary to the target biomolecule.

The third embodiment is not limited to disposing each of the biological material layers 91a to 91i directly on the surface of the first layer 13. In the example shown in FIG. 22, the silane coupling layers 81a, 81b, 81c, 81d, 81e, 81f, 81g are formed on the surface of the portion exposed through each of the plurality of wells 41a to 41i of the first layer 13. , 81h, 81i are arranged. In each of the silane coupling layer 81a~81i, as shown in FIG. 23, each of the methyl groups of the plurality of silane coupling agent (-CH ₃₎ or ethyl (-C ₂ H ₅₎ the first layer 13 It is chemically bonded to the hydroxyl (-OH) group on the surface by an acid-base reaction.

  Each of the silane coupling agents includes 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, N-2 (aminoethyl) 3 -Aminopropylmethyldimethoxysilane, N-2 (aminoethyl) 3-aminopropyltrimethoxysilane, N-2 (aminoethyl) 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltri Ethoxysilane or the like can be used.

On each of the silane coupling layers 81a to 81i, biological material layers 91a, 91b, 91c, 91d, 91e, 91f, 91g, 91h, 91i are arranged. In each of the biological material layer 91a~91i, for example, amino introduced active ester silane coupling agent in each of a plurality of biomolecules (-NH ₂₎ groups and amide (-NH-CO-) bond to Yes.

Alternatively, the silane coupling agent and the biomolecule may be bonded via a crosslinking agent. For example, receptors, ligands, antagonists, antibodies, amino of a lysine contained in the protein antigen such as (Lys) (-NH ₂₎ group, a carboxyl (-COOH) group of aspartic acid (Asp) and glutamic acid (Glu), tyrosine Functional groups such as (Tyr) phenol (-C ₆ H ₄ (OH)) group, histidine (His) imidazole (-C ₃ H ₃ N ₂ ) group, cysteine (Cys) thiol (-SH) group, etc. The amino group or epoxy group of the silane coupling agent may be bonded with a crosslinking agent. In the example shown in FIG. 24, the amino group of the silane coupling agent and the amino groups of antibodies 95a, 95b, and 95c are combined with disuccinimidyl suberate (DSS), a crosslinking agent that reacts with the amino group at both ends. ).

Other crosslinking agents include bissulfosuccinimidyl suberate (Bis [Sulfosuccinimidyl] suberate: BS ³ ), dimethyl suberimidate (HCl: DMS), and disuccicinimid, which react with amino groups at both ends. Disuccinimidyl glutarate (DSG), Loman's Reagent, 3, 3'-dithiobissulfosuccinimidyl propionate (DTSSP), ethylene glycol bis Succinimidyl succinate (Ethylene glycol bis [succinimidylsuccinate]: EGS), 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (1-Ethyl-3-[3 -Dimethylaminopropyl] carbodiimide Hydrochloride (EDC) can be used.

Furthermore, as cross-linking agents, m-maleimidobenzyl-N-hydroxysuccinimide ester (MBS), which reacts with amino group and thiol group, succinimidyl 4- [N-maleimidomethyl] -cyclohexane- 1-Carboxylate (Succinimidyl 4-[N-maleimidophenyl]-cyclohexane-1-carboxylate: SMCC), Succinimidyl 4-[p-Maleimidophenyl]-Butyrate (Succinimidyl 4-[p-maleimidophenyl]-buthrate: SMPB), N -Succinimidyl 3- (2-pyridyldithio) propionate (N-Succinimidyl 3- [2-pyridyldithio] propionate: SPDP), N- (γ-maleimidobutyryloxy) sulfosuccinimide ester (N- [γ-Maleimidobutyloxy] sulfosuccinimide ester: Sulfo-GMBS), Sulfosuccinimidyl 6-[3 '(2-Pyridyldithio)-propionamide] Hexanoate (Sulfosuccinimidyl 6-[3 '(2-pyridyldithio)-propionamide] hexanoate: Sulfo-LC-SPDP), m-Maleimidebenzoyl-N-hydoroxysulfo-succinimide ester: Sulfo-MBS) Sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfosuccinimidyl 4 [N-maleimidomethyl]-cyclohexane-1-carboxylate: Sulfo-SMCC), Sulfosuccinimidyl-4- ( p-Maleimidophenyl) butyrate (Sulfosuccinimidy 4-[p-malei
midophenyl]-butyrate: Sulfo-SMPB) can be used.

Each of the other wells 42a to 42i, 43a to 43i, 44a to 44g, 45a to 45g, 46a to 46g, 47a to 47i, 48a to 48i, and 49a to 49i shown in FIG. Since it is the same as that of FIG. Note that silica glass or the like can be used as the material for the first layer 13 shown in FIGS. 19 and 22 in addition to SiO ₂ . In this case, the semiconductor substrate 15 may be omitted. In addition to crystalline Si, a resin such as polytetrafluoroethylene or an insoluble epoxy resist can be used as the material for the second layer 11.

  On the top of the biochip substrate shown in FIGS. 18 to 24, as shown in FIG. 26 which is a cross-sectional view seen from the AA direction of FIGS. Be placed. As a material for the cover plate 25, quartz glass, acrylic resin, polycarbonate, or the like can be used. FIG. 27 shows a cross-sectional view seen from the direction AA when the cover plate 25 is arranged on the biochip substrate shown in FIG. When a test solution containing a biomolecule sample labeled with a fluorescent reagent such as Cy3 or Cy5 is injected from the opening 27 and the internal air is sucked from the opening 28, each of the wells 41a to 41i and the flow paths 31a to 31i Are sequentially filled with the test solution. Furthermore, the other plurality of wells 42a to 42i, 43a to 43i, 44a to 44g, 45a to 45g, 46a to 46g, 47a to 47i, 48a to 48i, 49a to 49i, and other plurality of flow paths shown in FIG. Each of 32a to 32i, 33a to 33i, 34a to 34g, 35a to 35g, 36a to 36g, 37a to 37i, 38a to 38i, and 39a to 39h are sequentially filled with the test solution.

  Therefore, when a target biomolecule that complementarily binds to a biomolecule fixed to the biomaterial layers 91a to 91i shown in FIG. 27 is included in the test solution, the target biomolecule is trapped in the biomaterial layers 91a to 91i. The Therefore, if the sample biomolecule contained in the test solution is labeled with a fluorescent reagent such as Cy3 in advance, after the test solution is aspirated, a plurality of wells 41a to 41i, 42a to 42i, 43a to 43i, 44a to 44g , 45a-45g, 46a-46g, 47a-47i, 48a-48i, 49a-49i, and multiple channels 31a-31i, 32a-32i, 33a-33i, 34a-34g, 35a-35g, 36a-36g, After washing each of 37a to 37i, 38a to 38i, and 39a to 39h, it is possible to confirm whether or not the target biomolecule is contained in the test solution by confirming the fluorescence reaction. If the diameter of each of the plurality of wells 41a to 41i, 42a to 42i, 43a to 43i, 44a to 44g, 45a to 45g, 46a to 46g, 47a to 47i, 48a to 48i, 49a to 49i is 600 mm or more, It is also possible to confirm the fluorescence reaction with the naked eye.

  As described above, the biochip substrate shown in FIGS. 18 to 27 includes a plurality of wells 41a to 41i, 42a to 42i, 43a to 43i, 44a to 44g, 45a to 45g, 46a to 46g, 47a to 47i, 48a to 48i. , 49a-49i, and a plurality of flow paths 31a-31i, 32a-32i, 33a-33i, 34a-34g, 35a-35g, 36a-36g, 37a-37i, 38a-38i, 39a-39h Therefore, when the cover plate 25 is disposed, high-precision alignment is not necessary. Also, the amount of test solution required is a plurality of wells 41a-41i, 42a-42i, 43a-43i, 44a-44g, 45a-45g, 46a-46g, 47a-47i, 48a-48i, 49a-49i, and Since it is sufficient to fill each of the plurality of flow paths 31a to 31i, 32a to 32i, 33a to 33i, 34a to 34g, 35a to 35g, 36a to 36g, 37a to 37i, 38a to 38i, 39a to 39h In addition, inspection of rare samples becomes easy. Also, since a plurality of flow paths 31a-31i, 32a-32i, 33a-33i, 34a-34g, 35a-35g, 36a-36g, 37a-37i, 38a-38i, 39a-39h are provided, Wells 41a-41i, 42a-42i, 43a-43i, 44a-44g, 45a-45g, 46a-46g, 47a-47i, 47a-47i, 48a-48i, 49a-49i are individually filled with a test solution using a device such as a spotter. This makes it possible to shorten the inspection time.

  Next, a method for manufacturing a biochip substrate according to the third embodiment will be described with reference to FIGS.

(a) First, as shown in FIG. 28, the semiconductor substrate 15, the first layer 13 made of SiO ₂ arranged on the semiconductor substrate 15, the second made of crystalline Si arranged on the first layer 13 An SOI (Silicon On Insulator) substrate having a natural oxide film 12 disposed on the layer 11 and the second layer 11 is prepared. Next, as shown in FIG. 29, a resist 21 is spin-coated on the natural oxide film 12.

 (b) A part of the resist 21 is removed by a lithography technique, and openings 51a, 51b, 51c, 51d, 51e, 51f, 51g, 51h, 51i are provided as shown in FIG. Next, a part of the natural oxide film 12 and the second layer 11 exposed from each of the openings 51a to 51i is selectively removed by anisotropic etching or the like, and a cross section viewed from the AA direction in FIGS. As shown in FIG. 32, the flow paths 31a to 31i, 32a to 32i, 33a to 33i, 34a to 34g, 35a to 35g, 36a to 36g, 37a to 37i, 38a to 38i, 39a to 39h The second layer 11 is formed.

 (c) As shown in FIG. 33, a resist 22 is spin-coated on the natural oxide film 12. Thereafter, a part of the resist 22 is removed by a lithography technique, and openings 61a, 61b, 61c, 61d, 61e, 61f, 61g, 61h, 61i are provided as shown in FIG. Next, the natural oxide film 12 and a part of the second layer 11 exposed from each of the openings 61a to 61i are selectively removed by anisotropic etching or the like until the first layer 13 is exposed, and FIG. Each of the wells 41a to 41i is formed in the second layer 11 as shown in FIG.

 (d) The SOI substrate is left in a stirred sodium hydroxide (NaOH) solution at room temperature for 2 hours. Here, the NaOH solution is a solution in which 98 g of NaOH, 294 ml of distilled water, and 392 ml of ethanol are mixed. By leaving it in the NaOH solution, the natural oxide film 12 on the surface of the second layer 11 is removed as shown in FIG. Further, as shown in FIG. 37, a plurality of hydroxyl (—OH) groups are introduced into the surface of the first layer 13 exposed from each of the wells 41a to 41i.

(e) A silane coupling agent having an epoxy in a functional group such as 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, or N-2 (aminoethyl) 3-aminopropyltriethoxy A first layer 13 expressing a silane coupling agent having an amine in a functional group such as silane, 3-aminopropyltrimethoxysilane, and 3-aminopropyltriethoxysilane from each of the wells 41a to 41i shown in FIG. Each of the silane coupling layers 81a, 81b, 81c, 81d, 81e, 81f, 81g, 81h, and 81i shown in FIG. 38 is formed. For example, when 3-aminopropyltrimethoxysilane is dropped, a plurality of amino (—NH ₂ ) groups are introduced on the surface of the first layer 13 as shown in FIG. Note that the unreacted OH group remaining on the surface of the first layer 13 is acetylated by capping with acetic anhydride and 1-methylimidazole (tetrahydrofuran solution), for example.

(f) First base nucleoside in which an active ester is introduced, the 5 ′ end is protected with a dimethoxytrityl (DMTr) group, and the amino group of a base such as adenine, cytosine, and guanine is protected as shown in FIG. Is dropped on the silane coupling layer 81a shown in FIG. The amino group of adenine and cytosine is protected with a benzoyl group. The amino group of guanine is protected with an isobutyl group. By dropping, the active ester of the first base nucleoside is reacted with the amino group (—NH ₂ ) of the silane coupling agent to form an amide bond (—NH—CO—) as shown in FIG. Next, the dimethoxytrityl (DMTr) group of the first base nucleoside was deprotected with 3% trichloroacetic acid / dichloromethane acidic solvent, and the 5 ′ hydroxyl (-OH) group was removed from the first base nucleoside as shown in FIG. To introduce.

 (g) Tetrazole and the nucleoside phosphoramidite shown in FIG. 43 are dropped onto the silane coupling layers 81a to 81i, and the N, N-- of the nucleoside phosphoramidite is added to the 5 ′ hydroxyl (-OH) group of the first base nucleoside. A diisopropylamino group is subjected to a condensation reaction. As shown in FIG. 44, the second base nucleoside is covalently bonded to the first base nucleoside by the condensation reaction. The unreacted 5'-hydroxyl group (-OH) group of the first base nucleoside is acetylated by treatment with acetic anhydride and 1-methylimidazole (tetrahydrofuran solution) and capped.

  (h) The phosphite triester bond generated by the condensation reaction is oxidized with iodine and water (pyridine-containing tetrahydrofuran solution) and converted into a more stable phosphate triester bond as shown in FIG. Thereafter, the second base dimethoxytrityl (DMTr) group is removed, and the condensation reaction with the nucleoside phosphoramidite is repeated until the target DNA chain length is reached as shown in FIG.

 (i) The cyanoethyl protecting group bonded to the phosphate group is eliminated by treatment with aqueous ammonia as shown in FIG. Further, the amino group-protected base shown in FIG. 40 is deprotected with aqueous ammonia as shown in FIG. Finally, the terminal dimethoxytrityl (DMTr) group is deprotected as shown in FIG. The steps shown in FIGS. 41 to 49 are also performed on each of the silane coupling layers 81b to 81i shown in FIG. 38 to form the biological material layers 91a to 91i as shown in FIG.

  According to the method for manufacturing a biochip substrate according to the third embodiment described above, as described in FIG. 36, by treating the SOI substrate with NaOH solution, only the surface of the first layer 13 is obtained. Hydroxyl (—OH) groups are introduced. When the second layer 11 is Si, the thickness of the natural oxide film 12 is usually 2 nm or less, but the natural oxide film 12 is removed by immersing in a NaOH solution at room temperature for 2 hours. Therefore, the silane coupling agent dropped in the subsequent step reacts only with the hydroxyl (—OH) group on the surface of the first layer 13 and does not bond to the surface of the second layer 11. Conventionally, when a silane coupling agent is bonded to the remaining natural oxide film 12, fluorescently labeled target biomolecules may also be bonded to the natural oxide film 12 to cause background noise. Especially in the micro assay, each of a plurality of wells 41a-41i, 41b-41i, 42a-42i, 43a-43i, 44a-44g, 45a-45g, 46a-46g, 47a-47i, 48a-48i, 49a-49i The target biomolecules are trapped only at the bottom of the substrate, and the target biomolecules are not trapped on the second layer 11, so that the appearance of technology that can improve the contrast of the fluorescence assay is expected. It was. In contrast, according to the method for manufacturing a biochip substrate according to the third embodiment, a hydroxyl (-OH) group is introduced only into the surface of the first layer 13, and at the same time the surface of the second layer 11 The natural oxide film 12 can be removed. Therefore, it is possible to manufacture a biochip substrate with extremely low background noise.

  FIG. 50 (a) shows that the wells 41a to 42i and 42a to 42i shown in FIG. 18 are sample DNAs labeled with a fluorescent reagent, which are complementary to the DNA immobilized on the biological material layer 91a shown in FIG. FIG. 2 shows an example of a micrograph when exposed to a test solution containing one having an array. FIG. 50 (b) shows a test solution including the sample DNA labeled with a fluorescent reagent in the conventional biochip substrate shown in FIGS. 62 to 67, which has a sequence complementary to the DNA immobilized on the substrate. It shows an example of a micrograph when exposed to. In the conventional substrate, it can be seen that the sample DNA is bound not only to the bottom of the well but also to other regions. Contamination is also observed at the bottom of the well. However, the biochip substrate according to the third embodiment can hybridize the target DNA only to the bottom of the well, as shown in FIG. 50 (a). In addition, the uniformity of fluorescence in the well is also realized.

  30 to 35, a plurality of flow paths 31a to 31i, 32a to 32i, 33a to 33i, 34a to 34g, 35a to 35g, 36a to 36g, 37a to 37i, 38a to 38i, 39a to 39h After forming each of the plurality of wells 41a-41i, 41b-41i, 42a-42i, 43a-43i, 44a-44g, 45a-45g, 46a-46g, 47a-47i, 48a-48i, 49a-49i The method of forming each was described above, but a plurality of wells 41a-41i, 41b-41i, 42a-42i, 43a-43i, 44a-44g, 45a-45g, 46a-46g, 47a-47i, 48a-48i , 49a to 49i, after forming a plurality of flow paths 31a to 31i, 32a to 32i, 33a to 33i, 34a to 34g, 35a to 35g, 36a to 36g, 37a to 37i, 38a to 38i, 39a to 39h Each may be formed. However, the depth of each of the plurality of flow paths 31a to 31i, 32a to 32i, 33a to 33i, 34a to 34g, 35a to 35g, 36a to 36g, 37a to 37i, 38a to 38i, 39a to 39h, Multiple wells 41a-41i, 41b-41i, 42a-42i, 43a-43i, 44a-44g, 45a-45g, 46a-46g, 47a-47i, 48a-48i, 49a-49i , It is better to form a plurality of flow paths 31a-31i, 32a-32i, 33a-33i, 34a-34g, 35a-35g, 36a-36g, 37a-37i, 38a-38i, 39a-39h first The uniform spin coating is easy.

  38 to 49, the method of forming the biological material layers 91a to 91i after forming the silane coupling layers 81a to 81i on the first layer 13 has been described. Of course, the biomolecule may be fixed to each of the hydroxyl groups without using the silane coupling layers 81a to 81i.

  28 to 49 show an example in which an SOI substrate is used, a biochip substrate may be manufactured using a bonded substrate of a glass substrate and a polytetrafluoroethylene substrate. That is, by using a glass substrate for the first layer 13 and a polytetrafluoroethylene substrate for the second layer 11, it is possible to manufacture a biochip substrate in the same manner as an SOI substrate.

(First modification)
The method for producing a biochip substrate is not limited to the method described above. Hereinafter, a method for manufacturing a biochip substrate using an epoxy negative resist will be described.

 (a) An epoxy negative first resist 111 is spin-coated on the surface of the first layer 115 made of glass or the like shown in FIG. Next, as shown in FIG. 52, the first resist 111 is exposed using a photomask 40 having light shielding patterns 140a, 140b, 140c,... Corresponding to wells to be formed.

 (b) As shown in FIG. 53, an epoxy negative second resist 211 is further spin-coated on the surface of the first resist 111 to form the second layer 11. Next, as shown in FIG. 54, the second resist 211 is exposed using a photomask 50 having a light shielding pattern 150 corresponding to the wells and flow paths to be formed.

 (c) The first resist 111 and the second resist 211 are baked. Note that the baking process renders each of the first resist 111 and the second resist 211 insoluble in a buffer solution or the like. Thereafter, development is performed with an alkali developer or the like, whereby the wells 41a to 41i and the flow paths 31a to 31i are formed in the second layer 11 as shown in FIG. Thereafter, the biochip substrate is completed by performing the method shown in FIGS.

(Other embodiments)
As described above, the present invention has been described according to the embodiment. However, it should not be understood that the description and drawings constituting a part of this disclosure limit the present invention. From this disclosure, various alternative embodiments, embodiments, and operation techniques should be apparent to those skilled in the art.

  For example, the biochip substrate according to the second modification shown in FIG. 57, which is a cross-sectional view seen from the AA direction of FIGS. 56 and 56, is different from the biochip substrate shown in FIGS. Each of the flow paths 31a to 31i, 32a to 32i, 33a to 33i, 34a to 34g, 35a to 35g, 36a to 36g, 37a to 37i, 38a to 38i, 39a to 39h are provided in the second layer 11. Absent. On the other hand, unlike the cover plate 25 shown in FIGS. 25 and 26, the cover plate 25 according to the second modified example shown in FIG. 59 which is a cross-sectional view seen from the BB direction of FIGS. When bonded to the biochip substrate shown in FIG. 57, a plurality of wells 41a to 41i, 41b to 41i, 42a to 42i, 43a to 43i, 44a to 44g, 45a to 45g, 46a to 46g, 47a to 47i , 48a to 48i and 49a to 49i.

  As shown in FIG. 60, which is a cross-sectional view when the cover plate 25 shown in FIG. 58 is bonded to the biochip substrate shown in FIG. 56, the test solution injected from the opening 27 is filled with the well 41a, Following the flow path 300, the wells 41b to 41i are sequentially filled. Therefore, as in the third embodiment, it is possible to reduce the amount of necessary test solution by using the biochip substrate shown in FIG. 56 and the cover plate 25 shown in FIG.

  Thus, it should be understood that the present invention includes various embodiments and the like not described herein. Therefore, the present invention is limited only by the invention specifying matters in the scope of claims reasonable from this disclosure.

1 is a first cross-sectional view of a biochip substrate according to a first embodiment of the present invention. It is 2nd sectional drawing of the board | substrate for biochips which concerns on the 1st Embodiment of this invention. It is a 1st process sectional view of the substrate for biochips concerning a 1st embodiment of the present invention. It is 2nd process sectional drawing of the board | substrate for biochips which concerns on the 1st Embodiment of this invention. It is 3rd process sectional drawing of the board | substrate for biochips which concerns on the 1st Embodiment of this invention. It is a 4th process sectional view of the substrate for biochips concerning a 1st embodiment of the present invention. It is 5th process sectional drawing of the board | substrate for biochips which concerns on the 1st Embodiment of this invention. It is 1st sectional drawing of the modification of the board | substrate for biochips which concerns on the 1st Embodiment of this invention. It is 2nd sectional drawing of the modification of the board | substrate for biochips which concerns on the 1st Embodiment of this invention. It is a 3rd sectional view of the modification of the substrate for biochips concerning a 1st embodiment of the present invention. It is 1st process sectional drawing of the biochip which concerns on the 1st Embodiment of this invention. It is 2nd process sectional drawing of the biochip which concerns on the 1st Embodiment of this invention. It is 3rd process sectional drawing of the biochip which concerns on the 1st Embodiment of this invention. It is a 4th process sectional view of the biochip concerning a 1st embodiment of the present invention. It is 5th process sectional drawing of the biochip which concerns on the 1st Embodiment of this invention. It is a photograph which shows the detection result of the biochip which concerns on the 2nd Embodiment of this invention. It is a photograph which shows the detection result of the conventional biochip. It is a top view of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is a 1st sectional view of the substrate for biochips concerning a 3rd embodiment of the present invention. It is a 2nd sectional view of the substrate for biochips concerning a 3rd embodiment of the present invention. It is a 1st expanded sectional view of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is a 3rd sectional view of the substrate for biochips concerning a 3rd embodiment of the present invention. It is a 2nd expanded sectional view of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is a 3rd expanded sectional view of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is a top view of the cover plate which concerns on the 3rd Embodiment of this invention. It is sectional drawing of the cover plate which concerns on the 3rd Embodiment of this invention. It is sectional drawing of the board | substrate for biochips and cover plate which concern on the 3rd Embodiment of this invention. It is 1st process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is 2nd process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is 3rd process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is a 1st process top view of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is 4th process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is 5th process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is 6th process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is 7th process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is 8th process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is 9th process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is 10th process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is 11th process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is a chemical formula which shows the base which concerns on the 3rd Embodiment of this invention. It is 12th process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is 13th process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is a chemical formula which shows the nucleoside phosphoramidite which concerns on the 3rd Embodiment of this invention. It is 14th process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is 15th process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is 16th process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is 17th process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is a chemical formula of the deprotected base which concerns on the 3rd Embodiment of this invention. It is 18th process sectional drawing of the board | substrate for biochips which concerns on the 3rd Embodiment of this invention. It is a photograph which shows the detection result of the biochip which concerns on the 3rd Embodiment of this invention. It is 1st process sectional drawing of the board | substrate for biochips which concerns on the 1st modification of the 3rd Embodiment of this invention. It is 2nd process sectional drawing of the board | substrate for biochips which concerns on the 1st modification of the 3rd Embodiment of this invention. It is 3rd process sectional drawing of the board | substrate for biochips which concerns on the 1st modification of the 3rd Embodiment of this invention. It is 4th process sectional drawing of the board | substrate for biochips which concerns on the 1st modification of the 3rd Embodiment of this invention. It is 5th process sectional drawing of the board | substrate for biochips which concerns on the 1st modification of the 3rd Embodiment of this invention. It is a top view of the board | substrate for biochips which concerns on other embodiment of this invention. It is sectional drawing of the board | substrate for biochips which concerns on other embodiment of this invention. It is a top view of the cover plate which concerns on other embodiment of this invention. It is sectional drawing of the cover plate which concerns on other embodiment of this invention. It is sectional drawing of the board | substrate for biochips and cover plate which concern on other embodiment of this invention. It is a perspective view of the conventional board | substrate for biochips. It is 1st process sectional drawing of the conventional board | substrate for biochips. It is 2nd process sectional drawing of the conventional board | substrate for biochips. It is 3rd process sectional drawing of the conventional board | substrate for biochips. It is a 4th process sectional view of the conventional board for biochips. It is 5th process sectional drawing of the conventional board | substrate for biochips. It is 6th process sectional drawing of the conventional board | substrate for biochips.

Explanation of symbols

A1, A2, A3, A4 ... Intermediate
A, B0, B1, B2, B3, B4 ... PCB
1, 15, 141… Semiconductor substrate
2, 2A, 13A, 23, 41a ~ 41i, 152, 162… Well
3, 132, 141a, 163… Si oxide layer
4, 14, 81a ~ 81i… Silane coupling layer
5a… Inactive spacer layer
5, 135… Spacer layer
6… Resin droplet
11 ... Second layer
12 ... Natural oxide film
13, 115 ... the first layer
16, 21, 22 ... resist
25… Cover plate
27, 28, 51a-51i, 61a-61i ... Opening
31a ~ 31i, 32a ~ 32i, 300 ... Flow path
40, 50… Photomask
91a-91i ... Biological material layer
95a, 95b, 95c… antibody
111 ... First resist
131… Semiconductor layer
140a ~ 140c ... Shading pattern
142 ... Non-reactive layer
150 ... Shading pattern
151 ... Glass plate
161… Plate
211 ... Second resist

Claims (2)

A silicon layer;
A silicon oxide layer disposed on the silicon layer;
A non-reactive layer disposed on the silicon oxide layer and provided with a plurality of wells reaching the silicon oxide layer;
A biological material disposed on the surface of the silicon oxide layer exposed through the well ,
The biochip substrate , wherein the non-reactive layer is made of at least crystalline silicon .   The biochip substrate according to claim 1, wherein the biochip substrate is manufactured from an SOI substrate.